AbstractThe use of the Cre‐loxP recombination system allows the conditional inactivation of genes in mice. The availability of transgenic mice in which the Cre recombinase expression is highly cell type specific is a prerequisite to successfully use this system. We previously have characterized regulatory regions of the mouse flk‐1 gene sufficient for endothelial cell‐specific expression of the LacZ reporter gene in transgenic mice. These regions were fused to the Cre recombinase gene, and transgenic mouse lines were generated. In the resulting flk‐1‐Cre transgenic mice, specificity of Cre activity was determined by cross‐breeding with the reporter mouse lines Rosa26R or CAG‐CAT‐LacZ. We examined double‐transgenic mice at different stages of embryonic development (E9.5–E16.5) and organs of adult animals by LacZ staining. Strong endothelium‐specific staining of most vascular beds was observed in embryos older than E11.5 in one or E13.5 in a second line. In addition, the neovasculature of experimental BFS‐1 tumors expressed the transgene. These lines will be valuable for the conditional inactivation of floxed target genes in endothelial cells of the embryonic vascular system. Developmental Dynamics 229:312–318, 2004. © 2004 Wiley‐Liss, Inc.